Dr. Raben and his colleagues have recently advanced the hypothesis that nuclear lipid metabolism is a component of nuclear signaling cascades which he defined as nuclear envelope signal transduction or NEST. The central, novel hypothesis to be tested in this proposal is that signals initiated at the plasma membrane are sent to the nucleus and trigger specific nuclear lipid metabolism as an integral component of plasma membrane-to-nucleus signal transduction cascades. Their studies indicate that nuclear phosphatidylcholine (PC) metabolism, and activation of PKC, are important components of NEST. The studies in this proposal examine nuclear PC metabolism and PKC activation induced in quiescent fibroblasts by alpha-thrombin, a potent mitogen. These studies represent the first series of experiments aimed at explicitly defining the molecular components involved in this novel signaling pathway. Defects in this cascade may lead to pathological condition including neoplasias. The studies are organized under three specific aims. The goal of Specific Aim I is to identify the GTP-binding protein(s) (GBP) responsible for coupling the alpha-thrombin receptor to nuclear PC metabolism and secondly, to determine whether MAPK (mitogen-activated protein kinase) is an intermediate component in this activation. The alpha-thrombin receptor couples to GBPs, and alpha- thrombin activates MAPK, a kinase implicated in nuclear signal transduction. In this aim, the investigators will test the hypothesis that the activation of a specific GBP(s) by the alpha-thrombin receptor results in the activation of a MAPK, leading to the activation of nuclear PC hydrolysis. GBP(s) coupled to the alpha-thrombin receptor and involved in induced nuclear PC hydrolysis will be identified by methods which will include antisense-mediated ablation of specific GBPs with a photoreactive GTP analog. The role of MAPK in these events will be determined by examining the ability of alpha-thrombin to activate nuclear PC hydrolysis in cells stably transfected with an inducible dominant- negative variant of MAPK. In Specific Aim II, they will identify, characterize and isolate the enzyme(s) responsible for nuclear PC hydrolysis involved in NEST. They will isolate, for the first time, eukaryotic PC hydrolyzing enzymes using new methods Dr. Raben has developed for expression cloning and using a new PC-coupled sepharose resin for affinity chromatography. In specific aim III, the investigators will explore the biological roles of nuclear DGs increases and PKC activation by identifying the nuclear PKC substrates using PKC inhibitors, in vitro phosphorylation, and sequence analyses.